Hiroyuki Azakami

Nucleotide sequence of the gene for monoamine oxidase (maoA) from Escherichia coli

Abstract We found that the structural gene for monoamine oxidase was located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that two amine oxidase genes are located in this region. The nucleotide sequence of one of the two genes was determined. The peptide sequence of the first 40 amino acids from the N terminus of monoamine oxidase purified from E. coli agrees with that deduced from the nucleotide sequence of the gene. The leader peptide extends over 30 amino acids. The nucleotide sequence of the gene and amino acid sequence of the predicted mature enzyme (M.W. 81,295) were highly homologous to those of the maoA K gene and monoamine oxidase from Klebsiella aerogenes , respectively. From these results and analysis of the enzyme activity, we concluded that the gene encodes for monoamine oxidase ( maoA E ). The tyrosyl residue, which may be converted to topa quinone in the E. coli enzyme, was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidases.

Effects of Er:YAG Laser Irradiation on Biofilm-forming Bacteria Associated with Endodontic Pathogens In Vitro

With the development of dental laser delivery systems that can enter into the root canals, it is possible to use Er:YAG lasers to remove the residual biofilm associated with infected root canals. We examined their effects against biofilms made of Actinomyces naeslundii, Enterococcus faecalis, Lactobacillus casei, Propionibacterium acnes, Fusobacterium nucleatum, Porphyromonas gingivalis, or Prevotella nigrescens in vitro. After Er:YAG laser irradiation with energy densities ranging between 0.38-0.98 J/cm(2), the biofilm samples on hydroxyapatite disks were quantitatively and morphologically evaluated. The Er:YAG laser was effective against biofilms of 6 of the bacterial species examined, with the exception of those formed by L. casei. After irradiation, the numbers of viable cells in the biofilms were significantly decreased, whereas atrophic changes in bacterial cells and reductions in biofilm cell density were seen morphologically. Er: YAG lasers might be suitable for clinical application as a suppressive and removal device of biofilms in endodontic treatments.

Enhanced secretion of hydrophobic peptide fused lysozyme by the introduction of N-glycosylation signal and the disruption of calnexin gene in Saccharomyces cerevisiae.

The insertion of a hydrophobic pentapeptide (Phe‐Phe‐Val‐Ala‐Pro) into the C‐terminus in hen egg white lysozyme by genetic modification resulted in an unstable structure which caused little secretion in a yeast expression system, although this modification is useful to enhance bactericidal action to Gram‐negative bacteria [Ibrahim et al. (1994) J. Biol. Chem. 269, 5059–5063]. To enhance the secretion of the unstable hydrophobic pentapeptide fused lysozymes (H5‐Lz), we attempted to introduce the signal sequence (Asn‐X‐Ser/Thr) of N‐linked glycosylation into lysozyme and to suppress the quality control of the unstable mutant in the yeast expression system. The polymannosyl hydrophobic fused lysozyme (H5/G49N‐Lz) having the N‐glycosylation signal sequence was expressed in the medium at 3.4 times that of unglycosylated lysozyme. Further, the secretion of the unstable mutant lysozyme was done in the Saccharomyces cerevisiae disrupted calnexin gene to avoid the degradation of the unstable mutant by the quality control. Although disruption of the calnexin gene did not lead to gross effects on the levels of growth of S. cerevisiae (W303‐1b), the secretion amount of H5/G49N‐Lz in calnexin disrupted S. cerevisiae was 2.5 times larger than that in wild type S. cerevisiae. These results suggest that the secretion of unstable glycosylated lysozyme (H5/G49N) was suppressed by the quality control function of calnexin and that the disruption of calnexin is effective to increase the secretion of unstable glycosylated protein.

Effects of the tea catechin epigallocatechin gallate on Porphyromonas gingivalis biofilms.

The aim of this study was to investigate the effects of tea catechin epigallocatechin gallate (EGCg) on established biofilms and biofilm formation by Porphyromonas gingivalis, a major pathogen of periodontal disease.

Antibiofilm Effects of Azithromycin and Erythromycin on Porphyromonas gingivalis

ABSTRACT Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections, such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis. Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ. We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibilities of adherent P. gingivalis strains 381, HW24D1, 6/26, and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX), and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC levels compared with those of the controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at levels over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICs; however, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels and could have future clinical application for oral biofilm infections, such as chronic marginal and periapical periodontitis.

P-domain and lectin site are involved in the chaperone function of Saccharomyces cerevisiae calnexin homologue

Cne1p, a calnexin homologue from Saccharomyces cerevisiae, has been shown to possess a conserved P‐domain and lectin site as mammalian calnexin. The effect of P‐domain and lectin site on the function of Cne1p was investigated in vitro using recombinant P‐domain, P‐domain deletion mutant of Cne1p, and lectin site mutant of Cne1ps (E181A and E398A) The binding of monoglucosylated oligosaccharide (G1M9) with Cne1p was clearly demonstrated using lectin site mutants. The P‐domain deletion mutant and the letcin site mutants partially decreased the ability to suppress the aggregation of citrate synthase (CS) and chicken egg yolk immunoglobulin at levels different from Cne1p. Furthermore, the P‐domain deletion mutant and the lectin site mutants decreased the ability to enhance the refolding of CS. These results suggest that the cooperation between the P‐domain and the lectin site are important for the complete function of Cne1p. Thus, we conclude that P‐domain in cooperation with the lectin site of Cne1p functions as a chaperone.

The Inhibitory Effects of Catechins on Biofilm Formation by the Periodontopathogenic Bacterium, Eikenella corrodens

Eikenella corrodens is a periodontopathogenic bacterium that forms biofilm even by itself. In this study, we investigated the inhibitory effects of catechins on E. corrodens biofilm formation. Biofilm formation was inhibited by the addition of 1 mM of the catechins with the pyrogallol-type B-ring and/or the galloyl group. The catechins with the galloyl group were effective at smaller doses than those with only the pyrogallol-type B-ring. An inhibitory effect was observed even when these catechins and gallic acid were added at sub-minimal inhibitory concentration (MIC) or at concentrations that showed no bactericidal effect. These results suggest that some catechins at sub-MIC might inhibit biofilm formation. No inhibitory effect of catechins at sub-MIC on biofilm formation was observed in the luxS deletion mutant. Our studies suggest that some species of catechins with the galloyl group affect autoinducer 2-mediated quorum sensing and thereby inhibit biofilm formation by E. corrodens.

Contribution of structural reversibility to the heat stability of the tropomyosin shrimp allergen.

Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS-PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment.

Expression, purification, and characterization of an unstable lysozyme mutant in Pichia pastoris

To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composition, induction time, and fermenter type were important factors for the expression of H5-lysozyme. Substantially active H5-lysozyme was secreted by induction with methanol when the prepro-sequence of alpha-factor was used as secretion signal sequence. The amount secreted was 422-fold greater than that observed with Saccharomyces cerevisiae. Recombinant H5-lysozyme was recovered and purified by cation-exchange chromatography directly from fermentation broth. The mutant lysozyme showed bactericidal activity against Gram-positive as well as Gram-negative bacteria.

Synergistic effects of antibiotics and an N-acyl homoserine lactone analog on Porphyromonas gingivalis biofilms.

Aims:  To investigate the effects of the combined application of an N‐acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease.